Experimental Biology and Medicine

The arachidonic acid products that form the family of ubiquitous compounds known as eicosanoids have been of interest to renal investigators for a number of years. Their contribution to normal renal physiology and role in renal dysfunction have only recently been identified and appreciated. In this Mini-review, we will attempt to cover some recent observations concerning the synthesis of eicosanoids, their actions on renal tubular epithelial ion transport mechanisms, and their possible role in renal function. Because of space limitations, it was not possible to review the entire family of the diverse group of arachidonic acid metabolites; therefore, an emphasis was placed on observations concerning the actions and role of renal prostanoids.

Eicosanoid Synthesis and Regulation

Prostaglandins have fascinated investigators ever since the initial discovery in the ′30s that a substance in human semen contracted the human uterus. Subsequently, the active material was identified as a lipid-soluble acid which von Euler named prostaglandin (1). However, it wasn't until the late ′70s that the actions of the prostaglandins were defined and many of this family of unique compounds isolated. Since then, a host of derivatives of the unsaturated lipid, arachidonic acid (C_{20:4 n-6}), have been shown to have a variety of biological activities that appear to modulate local cellular function. Prostaglandins belong to a family of compounds known as eicosanoids, which include not only derivatives of cyclo-oxygenase, but also the lipoxygenase products, the leukotrienes, lipoxins, and various hydroxy-fatty acids. More recently, another pathway for arachidonic acid metabolism was identified that involves the cytochrome P-450-dependent monooxygenases (2). This latter pathway has received heightened interest, particularly with respect to its possible role in the kidney (3).

Adrenal corticosteroids have diverse actions in the central nervous system (CNS). Glucocorticoids, the major adrenal corticosteroids, regulate the actions of several classical neurotransmitters and peptides, neuronal and glial proliferation and differentiation, neuronal survival, cognitive function, and behavior (1). Mineralocorticoids, e.g., aldosterone, regulate salt appetite and blood pressure (2, 3). As with other members of the steroid receptor superfamily, most actions of adrenal corticosteroids are mediated by intracellular receptors (4). Activated corticosteroid receptors bind to a glucocorticoid response element and interact with other transcription factors to regulate the expression of various genes (5–8). Two types of corticosteroid receptors have been described (7–9). A Type I receptor, which is identical to the classical mineralocorticoid receptor of the kidney, salivary glands, and colon, has a high affinity (K_D = 0.5 nM) and low capacity for endogenous glucocorticoids in the brain. It binds preferentially to corticosterone in the limbic brain, and presumably mediates the effects of circadian levels of circulating corticosterone on CNS function. In the cir-cumventricular regions and ventrolateral hypothalamus, the Type I receptor shows a preference for aldosterone, and may mediate the central effects of this mineralocorticoid on salt appetite and blood pressure. A lower affinity (K_D = 2.5–5 nM), high capacity Type II receptor, which is identical to the classical glucocorticoid receptor of the liver, has also been described in the CNS. It is thought to mediate the effects of stress levels of endogenous glucocorticoids and synthetic glucocorticoids, e.g., dexamethasone, on CNS function and central negative feedback regulation of plasma glucocorticoid levels.

Corticosteroid Receptor Immunoreactivity in the CNS

The availability of antibodies against Type I (10–12) and Type II (13–15) corticosteroid receptors has facilitated immunocytochemical studies on their distribution in various target tissues. Using MINREC anti-sera against peptides derived from the hinge and variable domains of the human renal Type I receptor cDNA (10, 11), and BUGR2 monoclonal antibody against rat liver Type II receptor (15), we have demonstrated the expression of both types of corticosteroid receptors in limbic, motor, sensory, and visceral neurons in the rat CNS (16–18). In most regions, the density of Type II-immunoreactive (ir) neurons is higher than Type I-ir neurons.

The present study was designed to investigate CI″ transport across rat ileal basolateral membranes. Basolateral membrane vesicles were prepared by a well-validated technique. The purity of the basolateral membrane vesicles was verified by marker enzyme studies and by studies of d-glucose and calcium uptake. Cl^- uptake was studied by a rapid filtration technique. Neither an outwardly directed pH gradient, nor a HCO₃ ⁻ gradient, or their combination could elicit any stimulation of Cl⁻ transport when compared with no gradient. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid at 5 mM concentration did not inhibit Cl⁻ uptake under gradient condition. Similarly, the presence of the combination of outwardly directed Na⁺ and HCO₃ ⁻ gradients did not stimulate Cl⁻ uptake compared with the combination of K⁺ and HCO₃ ⁻ gradients or no HCO₃ ⁻ gradient. This is in contrast to our results in the brush border membranes, where an outwardly directed pH gradient caused an increase in Cl⁻ uptake. Cl⁻ uptake was stimulated in the presence of combined Na⁺ and K⁺ gradient. Bumetanide at 0.1 mM concentration inhibited the initial rate of Cl⁻ uptake in the presence of combined Na⁺ and K⁺ gradients. Kinetic studies of bumetanide-sensitive Cl⁻ uptake showed a V_max of 5.6 ± 0.7 nmol/mg protein/5 sec and a K_m of 30 ± 8.7 mM. Cl⁻ uptake was stimulated by an inside positive membrane potential induced by the ionophore valinomycin in the setting of inwardly directed K⁺ gradient compared with voltage clamp condition. These studies demonstrate two processes for Cl⁻ transport across the rat ileal basolateral membrane: one is driven by an electrogenic diffusive process and the second is a bumetanide-sensitive Na⁺/K⁺/2 Cl⁻ process. Cl⁻ uptake is not enhanced by pH gradient, HCO₃ ⁻ gradient, their combination, or outwardly directed HCO₃ ⁻ and Na⁺ gradients. [P.S.E.B.M. 1992, Vol 201]

It has been reported that atrial natriuretic peptide (ANP) concentrations are elevated in pregnancy and further elevated in pregnancy-induced hypertension. Atrial stretch and volume expansion appear to be important stimuli for ANP release. During normal pregnancy, a striking change in hemodynamics occurs that may increase plasma ANP concentrations. ANP has potent natriuretic, diuretic, and smooth muscle relaxant activities. The biological effects of ANP during pregnancy may play an important role in the physiology and pathophyisology of pregnancy. Because of possible interactions during pregnancy due to secondary effects of maternal cardiovascular changes and physiological adaptation, the present study sought to evaluate and characterize the local effects of atriopeptin II on the uterine vascular bed of the nonpregnant sheep. Ewes with catheters in the femoral artery, femoral vein, and uterine artery and electromagnetic flow probes on the middle uterine arteries were monitored for blood pressure (BP), heart rate (HR), and uterine blood flow before and after the administration into the uterine artery of bolus injections of 2, 4, 20, and 40 × 10⁻⁹ M (5, 10, 50, and 100 μg) of the synthetic ANP (atriopeptin II). For comparison purposes, the effects of prostaglandin l₂ in doses of 1.2, 2.5, 12, and 25 × 10⁻⁸ M (5, 10, 50, and 100 μg), vasoactive intestinal polypeptide in doses of 3, 9, 30, 90, 300, and 900 × 10⁻¹¹ M (0.1, 0.3, 1, 3, 10, and 30 μg), and bradykinin in doses of 9.4, 28, 94, 280, 940, and 2800 × 10⁻¹¹ M (0.1, 0.3, 1, 3, 10, and 30 μg) were also tested. Appropriate vehicles were tested and found to be without effect.

All four compounds were found to be vasodilators of the nonpregnant uterine vasculature. ANP administered into the uterine artery decreased BP (87 ± 4 mm Hg to 79 ± 4 mm Hg with 50 μg [20 × 10⁻⁹ M]), increased HR (90 ± 5 bpm to 105 ± 4 bpm), and significantly increased uterine blood flow (from 14 ± 3 to 37 ± 4 ml/min with a dose of 100 μg [40 × 10⁻⁸ M, P < 0.05]). Prostaglandin l₂ failed to alter BP, but caused significant increases on HR (100 ± 4 to 124 ± 13 bpm, P < 0.05) and uterine blood flow (17 ± 4 to 73 ± 10 ml/min, P < 0.05). Vasoactive intestinal polypeptide caused a significant tachycardia (97 ± 10 to 158 ± 9 bpm, P < 0.05) at the highest dose, Bradykinin increased BP significantly at two of the highest doses. These data indicate that local intra-arterial bolus injections of ANP not only caused local uterine effects, but also had significant effects on BP and HR, which were qualitatively similar to those produced by vasoactive intestinal polypeptide. [P.S.E.B.M. 1992, Vol 201]

Feline intrapulmonary arteries (mean diameter, 0.9 mm) were equilibrated in Earle's solution at constant tension in a chamber bubbled with an hyperoxic gas mixture (30% oxygen, 5% carbon dioxide, balance nitrogen). The endothelium was removed from half the vessels by gentle rubbing. The isometric response to the addition of acetylcholine (1∗10⁻⁶ M) was dilator in the vessels with endothelium and constrictor in those without endothelium. Intermittent exposure to a hypoxic gas mixture (0% oxygen, 5% carbon dioxide, balance nitrogen) for 20 min with five repetitions demonstrated sustained constrictor responses in the presence or absence of endothelium. Endothelial cells are, therefore, not required for the mediation of hypoxic pulmonary vasoconstriction. [P.S.E.B.M. 1992, Vol 201]

Thyroxine has been shown in vitro to stimulate erythropoiesis by two mechanisms: a direct, β₂-adrenergic receptor-mediated stimulation of red cell precursors, and an indirect, erythropoietin-mediated mechanism. Clinical reports have suggested that excess thyroxine also exerts depressive effects on thrombocytopoiesis, but the most sensitive methods of assessing platelet production, i.e., percentage of ³⁵S incorporation into platelets and determination of megakaryocyte size and number, are not appropriate for analysis of platelet production in human patients. The purpose of this study was to use a mouse model to investigate the effects of the hyperthyroid state on erythropoiesis and thrombocytopoiesis, and to assess in vivo the two mechanisms by which thyroxine has been described to stimulate erythropoiesis in vitro. We found that thyroxine administration significantly depressed platelet production and stimulated erythropoiesis in mice. Both the D- and L-isomers of thyroxine in appropriate doses produced this depression of thrombocytopoiesis, and the effect was dose dependent for both isomers. Daily administration of thyroxine:increased blood volume; decreased the peripheral platelet count, total circulating platelet count and mass, percentage of ³⁵S incorporation into platelets, and megakaryocyte number and size; and concurrently increased indices of red cell production (packed cell volume, red blood cell count, total circulating red blood cell count and mass, and reticulocyte count). Additionally, propranolol, a nonspecific β-blocker, partially reversed the suppression of platelet production by L-thyroxine, lending credence to the assertion that the direct, β₂-adrenergic receptor-mediated stimulation of the erythroid cell line by thyroxine reported to exist in vitro may also be important in vivo. [P.S.E.B.M. 1992, Vol 201]

Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) are decreased and mean arterial pressure (MAP) and renal vascular resistance (RVR) are increased after unilateral release of bilateral ureteral obstruction (BUO) of 24 hr duration. An imbalance between vasoconstrictor and vasodilator substances may explain these hemodynamic changes. We examined the role of the cytochrome P-450 pathway in this setting. After unilateral release of BUO, GFR and ERPF (ml/min/kg body wt) were significantly lower in these rats than in sham-operated rats (SOR) 1.14 ± 0.09 vs 6.7 ± 0.5 and 3.09 ± 0.2 vs 23.5 ± 3.4, respectively). BUO rats had significantly higher MAP (mm Hg) and RVR (mm Hg/ml/min/kg body wt) than SOR (155 ± 5 vs 120 ± 1 and 29.1 ± 1.7 vs 3.2 ± 0.4, respectively). SOR given 3-methylcholanthrene and β-naphthoflavone to induce the cytochrome P-450 system had no significant changes in renal function, RVR, or MAP. SOR given ketoconazole to inhibit the cytochrome P-450 system had significantly lower GFR (4.8 ± 0.5) than temporal control rats without significant changes in ERPF (21.2 ± 4.6), MAP (127 ± 6), or RVR (4.2 ± 0.9). Rats with BUO given ketoconazole had lower but not significantly different GFR (0.84 ± .1) and ERPF (2.61 ± .4) than BUO controls. Values for MAP did not differ in BUO rats given ketoconazole versus BUO temporal controls. BUO rats given 3-methylcholanthrene and β-naphthoflavone had significantly higher GFR and ERPF (2.01 ± 0.24 and 6.66 ± 1.36, respectively) and significantly lower RVR (14.7 ± 3.9) than control rats with BUO; MAP was unchanged. Microsomal preparations from indomethacin-treated isolated kidneys obtained from BUO rats when compared with preparations obtained from SOR had significantly less activity of the P-450 cytochrome-dependent ω/ω-1 hydroxylase (103 ± 6 vs 130 ± 7 pmol hydroxyeicosatetraenoic acids produced per mg of protein/min, P < 0.02) and the P-450 cytochrome-dependent epoxygenase (11 ± 0.3 vs 30 ± 4 pmol lipoxyeicosatrienoic acids produced per mg of protein/min, P < 0.04). Indomethacin-treated microsomes prepared from kidneys of BUO rats converted significantly less ¹⁴C-arachidonic acid through the P-450-dependent hydroxylases (13.5 ± 0.8 vs 17.0 ± 0.1% of ¹⁴C-arachidonic acid converted to 19- and 20-hydroxyeicosatetraenoic acids, P < 0.02), and significantly less through the epoxygenases (1.4 ± 0.4 vs. 3.8 ± 0.5% of ¹⁴C-arachidonic acid converted to epoxyeicosatrienoic acids). The data suggest that a metabolite(s) of the cytochrome P-450 pathway modifies renal function in vivo in such a way that inhibition or activation of the cytochrome P-450 system is reflected in decreases or increases in GFR and ERPF in BUO rats without affecting mean arterial pressure. We conclude that the cytochrome P-450 pathway plays an important role in renal hemodynamics of normal rats and rats with BUO of 24 hr duration. [P.S.E.B.M. 1992, Vol 201]

The in vitro effect of lithium on lymphokine-activated killer cell (LAK) activity and its in vivo antitumor growth were observed. LAK activity was enhanced when LiCI was added during LAK cell induction, and this enhancement was observed both in human peripheral blood mononuclear cell and in mouse splenocytes used as LAK precursors. Cholera toxin, which can increase intracellular levels of cAMP, decreased LAK cell activity. However, lithium partially reversed this inhibitory effect, indicating that lithium increased LAK cell activity by decreasing cAMP levels. D-Sphingosine, an inhibitor of protein kinase C, and EGTA, a calcium chelator, both inhibited the LAK cell activity. However, their inhibitory effects could not be reversed by lithium because lithium was added in the culture in combination with one of these inhibitors during LAK cell induction. By using slot blot analysis, the effect of lithium on the expression of tumor necrosis factor-α mRNA of LAK cells was analyzed. Lithium increased the level of tumor necrosis factor-α mRNA when both lithium and interleukin 2 were added to induce LAK cells. The in vivo antitumor effect of lithium has also been studied. Using a mouse melanoma experimental model, the effect of lithium on tumor growth was also observed. Both lithium alone and interleukin 2/LAK had an antitumor effect, whereas the treatment of interleukin 2/LAK in combination with lithium had the strongest inhibitory effect on tumor growth, since this treatment resulted in reduction of tumor size and prolongation of survival in tumor-bearing mice. Therefore, it is hopeful that lithium can be used as a new immunomodulator for cancer immunotherapy and immune diseases. [P.S.E.B.M. 1992, Vol 201]

The present studies were designed to examine the regulation of Na⁺/H⁺ exchange activity by epidermal growth factor (EGF) in an in vitro system. Na⁺/H⁺ exchange activity was determined in brush-border membranes isolated from rat jejunal enterocytes incubated with epidermal growth factor and a number of second messengers. EGF at physiological concentrations stimulated Na⁺/H⁺ exchange activity without affecting vesicle size. The stimulation of Na⁺/H⁺ activity was the result of increasing V_max of Na⁺/H⁺ (6.0 ± 0.4 compared with 3.3 ± 0.27 nmol/mg protein/5 sec, P < 0.01). K_m values of the Na⁺/H⁺ exchanger in brush-border membrane from cells stimulated with EGF and controls were similar (16.0 ± 3.0 vs 13.0 ± 3.0, respectively). Na⁺/H⁺ activity was inhibited by phorbol esters, calmodulin, and cyclic AMP. The effects of EGF, calmodulin, cyclic AMP, and phorbol esters were dependent on ATP, because depleting the cells from ATP masked the effects on Na⁺/H⁺ exchange activity. The results suggest that EGF stimulates Na⁺/H⁺ exchange activity in the enterocytes. This stimulation is most likely not via activation of the phosphatidylinositol pathway. [P.S.E.B.M. 1992, Vol 201]

Growth hormone (GH) was measured in the sera of control, hypothyroid (thyroidectomized [Tx]) and GH-treated Tx rats and their fetuses on Days 19, 20, 21, and 22 of gestation and in their progenies on postnatal Days 1, 5, 30, and 75. Maternal endogenous serum GH increased dramatically between the 19th and 20th days of gestation and remained elevated through the 22nd day in control rats, but was depressed significantly in Tx and GH-treated Tx rats during this period. GH was not always detected in the sera of 19-day-old fetuses. On Day 20, GH was depressed in fetuses of Tx mothers as compared with those from controls or GH-treated Tx mothers. GH was elevated in sera of fetuses from GH-treated Tx rats over fetuses of control and Tx only rats on the 22nd day of gestation. In postnatal rats, those from GH-treated mothers continued to show elevated serum GH on Day 1 as compared with those from Tx only mothers. On postnatal Days 5 and 30, progenies of Tx mothers had significantly elevated GH as compared with progenies of control mothers. At 75 days of age, the GH levels of these progenies had normalized. We have shown previously that the hormonal secretions of the pituitary-thyroid axis are badly disrupted in the progenies of Tx and GH-treated Tx mothers and that even as adults these animals have tissue (brain and liver) deficits of active thyroid hormones. Although the onset of GH secretion is mildly delayed in fetuses of Tx but not GH-treated Tx mothers, the serum GH levels of both groups of progenies are elevated during most of the neonatal period through the time of puberty. It is, therefore, concluded that GH in the absence of adequate levels of thyroid hormones is ineffective in preventing many of the learning and memory deficits induced in the progenies of Tx mothers. [P.S.E.B.M. 1992, Vol 201]